Halohydrin dehalogenase (“HHDH”), also named halohydrin hydrogen-halide-lyase or halohydrin epoxidase, [EC4.5.1] catalyzes the interconversion of 1,2-halohydrins and the corresponding 1,2-epoxides:
U.S. Pat. No. 4,284,723 describes the use of a halohydrin epoxidase for the production of propylene oxide. U.S. Pat. Nos. 5,166,061 and 5,210,031 describe the use of this enzyme activity for the conversion of 1,3-dichloropropanol (DCP) and epichlorohydrin (ECH) respectively to 4-chloro-3-hydroxybutyronitrile (CHBN). HHDH enzymes from Agrobacterium radiobacter and Corynebacterium have been characterized on a broad range of halogenated substrates (Van Hylckama Vlieg et al., J. Bacteriol. (2001) 183:5058-5066; Nakamura et al., Appl. Environ. Microbiol. (1994) 60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992) 36:478-482).
HHDH also catalyzes the ring opening of epoxides with nucleophiles other than chloride or bromide. It has been demonstrated that azide (N3−), nitrite (NO2−) and cyanide (CN−) can replace chloride in the opening of epoxides (see Nakamura et al., Biochem. Biophys Res. Comm. (1991) 180:124-130; Nakamura et al., Tetrahedron (1994) 50: 11821-11826; Lutje Spelberg et al., Org. Lett. (2001) 3:41-43; Lutje Spelberg et al., Tetrahedron Assym. (2002) 13:1083):
Nakamura et al. (Tetrahedron (1994) 50: 11821-11826) describe the use of HHDH for the direct conversion of DCP to chloro-3-hydroxy-butyronitrile (CHBN) through epichlorohydrin (ECH) as the intermediate:

Some halohydrin dehalogenases have been characterized. For example, HHDH from A. radiobacter AD1 is a homotetramer of 28 kD subunits. Corynebacterium sp. N-1074 produces two HHDH enzymes, one of which is composed of 28 kD subunits (Ia), while the other is composed of related subunits of 35 and/or 32 kD (Ib). HHDH from some sources is easily inactivated under oxidizing conditions in a process that leads to dissociation of the subunits, has a pH optimum from pH 8 to 9 and an optimal temperature of 50° C. (Tang, Enz. Microbial Technol. (2002) 30:251-258; Swanson, Curr. Opin. Biotechnol. (1999) 10:365-369). The optimal pH for HHDH catalyzed epoxide formation has been reported as 8.0 to 9.0 and the optimal temperature in the range of from 45° C. to 55° C. (Van Hylckama Vlieg et al., J. Bacteriol. (2001) 183:5058-5066; Nakamura et al., Appl. Environ. Microbiol. (1994) 60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992) 36:478-482). The optimal pH for the reverse reaction, ring opening by chloride, has been reported for the two Corynebacterium sp. N-1074 enzymes and is 7.4 (Ia) or 5 (Ib). Site directed mutagenesis studies on the A. radiobacter AD1 HHDH indicated that oxidative inactivation is due to disruption of the quartenary structure of the enzyme by oxidation of cysteine residues (Tang et al., Enz. Microbial Technol. (2002) 30:251-258).
Purified HHDH enzymes from different sources exhibit specific activities on DCP ranging from 146 U/mg (Ib) to 2.75 U/mg (Ia) (Nakamura et al., Appl. Environ. Microbiol. 1994 60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992) 36:478-482). The high activity of the lb enzyme is accompanied by a high enantioselectivity to produce R-ECH from DCP, while the Ia enzyme produces racemic ECH.
HHDH encoding genes have been identified in Agrobacterium radiobacter AD1 (hheC), Agrobacterium tumefaciens (halB), Corynebacterium sp (hheA encoding Ia and hheB encoding lb), Arthrobacter sp. (hheAAD2), and Mycobacterium sp. GP1 (hheBGP1). All enzymes have been functionally expressed in E. coli. 
It is highly desirable for commercial applications of HHDH that the enzyme exhibits high volumetric productivity, that reactions run to completion in a relatively short period of time, with a high final product concentration, with high enanantioselectivity, and that no chemical side products are formed. These characteristics of a process can generally be used to define the broad characteristics of the enzyme: low Km for the substrate(s), high process stability, high specific activity, no substrate and product inhibition under conditions where chemical reactions are not proceeding. Currently available HHDH enzymes do not fulfill all of these criteria. For instance, the conversion on 1,2-epoxybutane and cyanide to 3-hydroxyvaleronitrile by HHDH proceeds at a maximum rate of 3 mmol/hr and this rate is sustained for only 10 minutes (Nakamura et al., Biochem. Biophys Res. Comm. (1991) 180:124-130). Conversion of DCP and ECH to 4-chloro-3-hydroxybutyronitrile (CHBN) is also limited to rates of 2-3 mmol/hr (Nakamura, U.S. Pat. Nos. 5,166,061 and 5,210,031). An in depth analysis of the ECH to CHBN conversion reveals that while the hheB encoded HHDH-Ib enzyme has high activity, high productivity is maintained for only 20 min after which further conversion occurs at a rate that is at least 50-fold slower, with the overall conversion at just over 60% (Nakamura et al. Tetrahedron (1994) 50: 11821-11826). The direct conversion of DCP, via ECH to CHBN proceeds at a reduced rate and results in a 65.3% yield. Thus, HHDH as described in the literature does not meet the desired criteria for a catalyst in commercial applications.
Accordingly, new halohydrin dehalogenases would be highly desirable.